Lentiviral–Mediated Gene Transfer of MYO7A in MYO7A Null RPE Cells

Abstract

Abstract: : Purpose: Mutations in the unconventional myosin VIIa cause Usher syndrome 1B. Shaker1 mice have mutations in the orthologous gene, and have a number of defects specific to the retinal pigmented epithelium (RPE). Ingested rod outer segments (ROS) are mislocalized and degraded slower than in control cells, and melanosomes have altered transport dynamics. Here, we investigated the ability of an HIV–1 lentiviral vector expressing human MYO7A to infect MYO7A–null RPE cells, and to rescue these mutant phenotypes. Methods: Primary RPE cells were cultured from MYO7A null mutant and heterozygous control mice. Mutant cells were infected with HIV–1 vector encoding the human MYO7A cDNA, either under the control of a full CMV promoter (LV.MYO7A.Ad) or a partial CMV promoter plus the MYO7A enhancer region (LV.MYO7A.Bd). Control mutant cells were either mock–infected or infected with HIV–1 vector encoding EGFP (LV.CIG). Seven days after infection, cells were screened for phagocytic ability using an ROS phagocytosis assay, and for melanosome trafficking using time–lapse microscopy and quantitative motion analysis. Results: Mutant RPE cells infected with LV.MYO7A.Bd vector showed >95% efficiency of infection after 7 days, with expression levels of MYO7A comparable to control cells and little evidence of cytotoxicity. In contrast, infection with LV.MYO7A.Ad resulted in over–expression and cell death, highlighting the need to control expression levels. Mutant RPE cells infected with LV.MYO7A.Bd showed rescue of the ROS degradation defect and of the melanosome trafficking defects, with ROS degradation kinetics and melanosome dynamics comparable to control cells analyzed in parallel. Mock–infected and LV.CIG infected cells showed no evidence of phenotypic rescue. Conclusions: We have found high efficiency, non–toxic expression of human MYO7A in MYO7A–null mouse RPE cells, using a lentiviral vector, with correction of both the ROS degradation and melanosome trafficking defects. Both of these defects are likely to contribute to the retinal degradation in Usher 1B, and rescue of these phenotypes in a cell culture system provides a proof of principle for treatment of this disorder in vivo using lentiviral mediated gene transfer.

Keywords: gene transfer/gene therapy • retinal degenerations: cell biology • retinal pigment epithelium